Hypertension is one of the most important public health problems in developed countries. It affects more than 60 million people in the United States alone. The disease, which is defined as an elevation of arterial blood pressure, results in secondary organ damage and a reduced lifespan. It occurs in two forms. Essential hypertension, also called primary hypertension, accounts for 95% of all cases, and is of unknown cause. Secondary hypertension, which constitutes the remaining 5% of the cases found, is associated with known causes, such as primary hyperaldosterism, renal disease, or coarctation of the aorta.
While no direct cause for the majority of hypertension cases have been found, some of the metabolic factors which regulate blood pressure are known. Notable among these are the angiotensin and kallikrein systems, which control vasoconstriction and dilation, and the salt-regulating system, which controls the levels of sodium ion in the bloodstream. The angiotensin system contains a number of critical proteins, prominent among which is renin, a key enzyme in the production of an octapeptide, angiotensin-II, which acts directly to constrict vascular smooth muscles and which also induces the release of aldosterone from the adrenal cortex. The proteins of the kallikrein system influence the level of kinin, a vasodilator. The levels of sodium ion are, on the other hand, regulated by an atrial natriuretic peptide factor (ANF). Renin, kallikrein, and ANF are human proteins of determined amino acid sequence. The genes encoding these proteins have been cloned and expressed. See U.S. Ser. Nos. 616,488, filed 1 June 1984, 719,414, filed 3 Apr. 1985, and 790,813, filed 24 Oct. 1985, all assigned to the same assignee and incorporated herein by reference.
There is, at present, no predictive diagnosis for hypertension, nor is there any accurate diagnosis for the various causes of hypertension. It is singularly important to have a predictive tool in the case of this disease, as various preventive therapies, based, for example, on control of the above-mentioned factors, are available. In addition, in relatively benign cases, even regulation of diet, lifestyle, or other noninvasive practices are effective in minimizing risk.
A technique that inherently offers the advantages of early detection, if its results can be effectively correlated with the disorder to be assessed, is genetic analysis. Since the genomic characteristics of an individual are basically determined, it is assumed, at conception, genetic aberrations which are indicia of later metabolic disorders are an ideal early diagnosis tool. Genetic testing can be routinely conducted using present methodology, as early as the seventh week of fetal life. In adults and children, it can be accomplished using a small blood sample. Over the last ten years, the availability of restriction enzymes and DNA probing techniques has made possible the use of "restriction fragment length polymorphisms" (RFLPs) in such diagnosis. Using the, by now, well established Southern blot hybridization technique (Southern, E., J Mol Biol (1975) 98:503-517), it has been possible successfully to diagnose sickle cell anemia (Kan, Y. W., et al, Proc Natl Acad Sci (USA) (1978) 75:5631); .beta.-thalassemia (Antonarakis, S. E., et al, Proc Natl Acad Sci (USA) (1983) 79:137); type II diabetes (Rotwein, P., et al, Science (1981) 213:1117); familial growth hormone deficiency (Phillips, J. A., III, Banbury Report 14, Cold Spring Harbor Laboratory (1983) pp 305-315); phenylketonuria (Woo, S. L. C., et al, Nature (1983) 306:151); Huntington's disease (Gusella, J. F., et al, Nature (1983) 306:234); and hemophilia B (Gianelli, et al, Lancet (1984) i:239, Grunenbaum, et al, J Clin Invest (1984) 73:1491).
The use of genetic detection systems is particularly appropriate in predicting the incidence of hypertension for an individual since it appears that genetic, rather than environmental, factors are in large part responsible for the onset of the disease. This has been shown in both animal and human studies, including of blood pressures which exist as patterns or "aggregations" within families. Adopted children do not demonstrate familial aggregation of blood pressures. Therefore, an assessment of the genome of the subject provides fertile ground for assessment of risk.
All of the successful predictive tests listed above are grounded in identification of a particular polymorphism or polymorphisms which correlates with the disease or disorder in question. It has been calculated that the number of polymorphisms expected in the human genome should be of the order of 10.sup.7, based on an assumed probability of 0.005 for a given nucleotide to be polymorphic; this number being inferred from studies of the human growth hormone, .alpha.I-antitrypsin and .beta.-like globin gene cluster loci (Jeffreys, A. J., Cell (1979) 18:1-10; Oster, H., et al, Am J Hum Gen (1984) 36(suppl) 150S). The challenge is to determine which of these over ten million polymorphisms is associated with a particular disorder, and to devise a procedure to detect it.
The present invention uses the gene loci associated with the production of renin, of ANF, or of kallikrein as sites for the occurrence of polymorphisms associated with hypertension.
The availability of the appropriate DNA sequences has made this a feasible approach. Human renin cDNA and genomic DNA have been described by Hardmann, J. A., et al, DNA (1984) 3:457; Imai, T., et al, Proc Natl Acad Sci (USA) (1983) 80: 7405; Hobart, P. M., et al, Proc Natl Acad Sci (USA) (1984) 81: 5026; Miyazaki, H., et al, Proc Natl Acad Sci (USA) (1984) 81: 5999. DNA encoding human ANF has been described by Greenberg, B. D., et al, Nature (1984) 312:656-658; Yamanaka, N., et al, Nature (1984) 309: 719; Maki, M., et al, Nature (1984) 309: 722; Oikawa, S., et al, Nature (1984) 309: 724; Seidman, C. E., et al, Science (1984) 225: 324; Zivin, R. A., et al, Proc Natl Acad Sci (USA) (1984) 81: 6325. The cDNA sequence encoding human kallikrein is disclosed in U.S. Ser. No. 790,813, filed 24 Oct. 1985 (supra).
The present invention provides a number of polymorphic sites in the above-mentioned gene regions which are useful both alone and in combination in predicting the incidence of hypertension by means of a simple blood test.